Project Summary Endogenous protein inhibitors of neovascularization (NV) and vascular leakage have great potential as therapeutic agents for retinal and choroidal vascular diseases, but thus far that potential has not been realized partly because large proteins present challenges for manufacturing, maintenance of appropriate folding, and penetration into tissues. We have developed a collagen IV-derived 20-mer peptide mimetic, AXT107 that strongly suppresses ocular neovascularization and vascular leakage. Intravitreous injection of 1g of AXT107 in mice has comparable activity to 40g of aflibercept and when combined, activity is greater than with either alone. In rabbit eyes, 50g AXT107 suppresses vascular leakage longer than 500g aflibercept because it self-assembles into a gel depot in the vitreous cavity that provides sustained delivery to the retina. The goal of this proposal is to elucidate the mechanism of action of AXT107 and the mechanism by which it self-assembles into a gel depot which provides prolonged activity. Preliminary data show that AXT107 binds ?v?3 which disrupts signaling through VEGFR2. AXT107 also disrupts signaling through platelet-derived growth factor receptor ? (PDGFR?), c-Met, and insulin-like growth factor-1 receptor; we will test the hypothesis that these effects are also a consequence of ?v?3 binding. AXT107 binds ?5?1 and activates Tie2 in the presence of Angiopoietin 2 (Angpt2). We will test the hypothesis that AXT107 binding dissociates ?5?1 into monomers, thereby eliminating ?5?1-induced constraint of Tie2 allowing it to translocate to cell junctions and form multimers that are activated by Angpt2 increasing junctional integrity. In mice with oxygen-induced ischemic retinopathy (OIR), we will test the hypothesis that high levels of Angpt2 which reduce the effects of the VEGF antagonist aflibercept, enhance the antiangiogenic and anti-permeability effects of AXT107 because in its presence Angpt2 is converted from a Tie2 antagonist to a Tie2 agonist. We will also test the hypothesis that AXT107 is superior to aflibercept + anti-Angpt2 because it does not depend solely on endogenous Angpt1 for Tie2 activation. The mechanism of AXT107 assembly and disassembly, and its effect on pharmacokinetics will be investigated. These studies will advance the development of AXT107 a novel therapeutic that combines two important activities, VEGF suppression and Tie2 activation, with prolonged duration of action, and thus addresses unmet medical needs for retinal and choroidal vascular diseases.